靶向VEGFR2基因的siRNA分子血清稳定性及其基因沉默效应

Construction of small interfering RNA targeting mouse vascular endothelial growth factor receptor-2:its serum stability and gene silencing efficiency in vitro

目的设计并合成抗小鼠VEGFR2基因的siRNA分子(siVEGFR2),研究其血清稳定性和基因沉默效应,以便进一步研究经组织靶向性结构修饰后该siRNA分子的特性。方法设计并合成抗小鼠VEGFR2基因的siRNA分子,在37℃条件下与新鲜血清共孵育24h,不同时间点后取样电泳观察其完整性;用脂质体介导转染体外培养的MS1细胞,通过半定量RT-PCR分析基因沉默效果。结果随着孵育时间延长,未经修饰的siVEGFR2分子在血清中逐渐降解;经siVEGFR2转染的MS1细胞中,VEGFR2mRNA的表达水平明显下降,与空白转染组和对照siRNA转染组相比有显著差异(P<0.001),而空白转染组和对照siRNA转染组间比较无显著性差异(P=0.157)。结论裸siVEGFR2分子在血清中容易降解,siVEGFR2能特异性沉默靶基因的表达,为下一步体内应用siVEGFR2分子时稳定性修饰的必要性提供了理论依据。

Objective To construct a small interfering RNA （siRNA） targeting mouse vascular endothelial growth factor receptor-2 （VEGFR2） and study its serum stability and gene silencing efficiency in vitro. Methods The synthesized siRNA targeting VEGFR2 （siVEGFR2） diluted in RNase-free water was mixed at a 1：1 ratio with fresh serum and incubated at 37 ℃. Gel electrophoresis was performed to determine the integrity of siVEGFR2 incubated for different time lengths. Oligofectamine 2000 was used to mediate siVEGFR2 transfection of MS 1 cells, and semi,quantitative RT-PCR was used to evaluate VEGFR2 gene silencing effect induced by the siRNA. Results The naked siRNA incubated in serum underwent gradual degradation with prolonged incubation time and became virtually undetectable after 24 h. Transfection of MS 1 cells with siVEGFR2 significantly down-regulated the expression of VEGFR2 mRNA in comparison with the blank group and control siRNA transfection group （P〈0.001）, while no significant difference was found in VEGFR2 mRNA levels between the latter two groups （P=-0.157）. Conclusion The naked siRNA is unstable in serum and not suitable for direct administration in vivo. The designed siRNA can effectively silence the VEGFR2 gene expression in MSI cells in vitro.